Fluorescent compounds are widely used in biological applications in which a highly sensitive detection reagent is desirable. In particular, the detection and quantification of calcium ion (Ca2+) levels in biological systems has become an important area of investigation in biological and medical research. For example, the measurement of calcium ions inside live cells using fluorescent indicators provides real-time and end-point readout in a variety of biological investigative techniques and in high throughput screening (HTS) of drug candidates.
Numerous natural and synthetic materials are known to selectively or non-selectively bind to or chelate metal ions. Ion chelators are commonly used in solution for in vivo control of ionic concentrations and detoxification of excess metals, and as in vitro buffers. When bound to a fluorophore, ion chelators can often be used as optical indicators in metal ion analysis. Certain types of metal ion (e.g., Ca2+) indicators utilize a chelating group in conjunction with a covalently attached fluorophore. One commonly used calcium ion chelating group is the tetracarboxylate chelating group based upon the structure of 1,2-bis-2-aminophenoxyethane-N,N,N′,N′-tetraacetic acid (BAPTA). Upon formation of the BAPTA chelate, the fluorescence properties of the attached fluorophore is affected in some measurable way (e.g., emission is enhanced or decreased or the wavelength of excitation or emission is altered). Ca2+ concentration can be determined using the measured fluorescence properties of a sample containing the indicator in conjunction with the dissociation constant for a specific indicator-Ca2+ complex.
Certain types of fluorescence-based ion indicators respond to metal ion binding by changes in the fluorescence excitation and/or emission wavelength maxima. Indicators having such fluorescence properties can be used as ratiometric indicators. Ratiometric indicators are widely used in imaging applications and in flow cytometry to determine intracellular metal ion (e.g., Ca2+) levels. Ratiometric measurements involve calculating a ratio between the excitation or emission intensity at two different wavelengths. Ratioing can reduce the effects of uneven dye loading, leakage of dye, photobleaching, and problems associated with measuring metal ions in cells of unequal thickness. Concentration measurements with ratiometric indicators generally are more convenient and accurate than measurements using intensity-based indicators.
Despite the abundance of fluorescent metal ion indicators (e.g., Ca2+ indicators), known indicators suffer from various drawbacks. For example, many indicators have fluorescence properties in the ultraviolet region. UV excitable indicators require the use of specialized quartz optics and detection is complicated by interference from the environment (for example, due to the natural fluorescence many biological materials). Certain indicators exhibit an increase in emission intensity only upon binding to calcium ions. Indicators exhibiting only an emission intensity increase indicator frequently display no wavelength shift in either the excitation or emission spectrum upon binding, which makes it difficult to measure the concentration of metal ions, such as Ca2+, using conventional ratiometric techniques. In addition, many ratiometric fluorescent metal ion indicators are limited to non-aqueous solutions due to insolubility or low quantum yield of the indicator in water or have metal ion binding affinities outside of physiologically relevant ranges. Despite continued research efforts, the assortment of the ratiometric fluorescent ion indicators available commercially is limited to two classes of calcium and magnesium indicators (Fura (excitation ratiometric), and Indo (emission-ratiometric) indicators). Examples of excitation ratiometric indicators include the sodium indicator SBFI and the potassium indicator PBFI. Members of the Fura and Indo classes of indicators can exhibit excitation ratiometry in the 300-335 nm region of the electromagnetic spectrum. However, the only available longer-wavelength excitation ratiometric calcium indicator, i.e. BTC, has a low calcium ion affinity (Kd˜7000 nM compared to ˜200 nM for Indo and Fura), which limits its utility as a metal ion sensor.
Thus, there exists a need for fluorescent ratiometric indicators (in particular, emission ratiometric indicators) that are useful in the desirable visible wavelength range and are compatible with the aqueous systems commonly utilized in biological applications.